The present invention relates to methods for producing polypeptides of interest in toxin-deficient Aspergillus mutant cells. The present invention also relates to mutants of Aspergillus cells and to methods for obtaining said mutant cells.
The use of recombinant host cells in the expression of heterologous polypeptides has in recent years greatly simplified the production of large quantities of commercially valuable polypeptides, such as industrially important enzymes and secondary metabolites, which otherwise are obtainable only at lower quantities or by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given polypeptide, including eubacterial and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce adequate yields of the polypeptide with the desired composition and conformation, but, to a large extent, may also be governed by the intended end use of the protein.
One problem encountered in connection with the use of certain host systems is the production of mycotoxins. A number of fungi, which are used as host cells in the production of polypeptides of interest possesses genes encoding enzymes involved in the biosynthesis of various toxins. For example, cyclopiazonic acid, kojic acid, 3-nitropropionic acid and aflatoxins are known toxins, which are formed in, e.g., Aspergillus flavus. Similarly, trichothecenes are formed in a number of fungi, e.g., in Fusarium sp. such as Fusarium venenatum and in Trichoderma. A detailed overview of the formation of toxins in different fungi can be found in Handbook of Toxic Fungal Metabolites, Richard J Cole and Richard H. Cox, Academic Press, 1981.
The formation of such toxins during the fermentation of the polypeptides of interest is highly undesirable as they may present a health hazard to both operators, customers and the environment.
Consequently, a lot of effort is spent ensuring that such toxins are not formed under the conditions used in the relevant productions in levels considered to affect the health. This is mainly done by an extensive analytical program where the toxins are analysed directly and by bioassays and/or feeding studies. In many cases these extensive programs are carried out on every single production batch affecting both production costs and the time before the products can be sold.
Cyclopiazonic acid (hereinafter also referred to as xe2x80x9cCPAxe2x80x9d) is a weak acid (pKa: 3.5) and precipitates under acidic conditions. It forms metal chelates, which can be split by dilute acid. It is quite toxic leading among other things to degenerative changes and necrosis in many organs, and selectively inhibits Ca2+-ATPase. CPA is produced in an xcex1- and xcex2-form, the xcex2-form being a precursor for the xcex1-form. CPA is produced by Aspergilli but also by other fungi, such as Penicilli.
Kojic acid (hereinafter also referred to as xe2x80x9cKAxe2x80x9d) is produced by a large number of Aspergilli but also by other fungi, such as Penicilli, and even by some bacteria. It is weakly alkaline (pKa: 7.9; phenolic group), and forms complexes with many metal ions. It has antimicrobial activity and is weakly toxic to animals. It is a precursor for a number of synthetic compounds like insecticides, dyes, etc.
3-Nitropropionic acid (hereinafter also referred to as xe2x80x9c3-NPAxe2x80x9d) is a natural nitro compound. It is produced by some fungi, especially Aspergilli (A. flavus, A. wentii) and Penicilli (P. atroventum). It has been reported in a few bacteria. The acid or its esters are also found in some plants. It is rather toxic in itself leading to, e.g., anemia. Also, it may be partly converted to another toxic compound nitrite in the gastrointestinal tract. 3-Nitropropionic acid affects Krebs cycle by inhibiting succinate dehydrogenase irreversibly and isocitrate lyase, fumarase and aspartase reversibly.
Aflatoxins are extremely biologically active, secondary metabolites produced by the fungi Aspergillus flavus Link ex. Fries and Aspergillus parasiticus Speare; see R. W. Detroy et al. xe2x80x9cAflatoxin and related compoundsxe2x80x9d, In Microbial Toxins, Vol. 6 (A. Ciegler, S. Kadis, and S. J. Ajl, Eds.), Academic, New York, 1971, pp. 3-178. The major aflatoxins are B1, B2, G1, and G2. The metabolites, particularly aflatoxin B1, are not only toxic to animals as well as humans but are also the most carcinogenic of all known natural compounds.
Malformins and ochratoxins are produced by A. niger. 
By eliminating or reducing the ability of the host organisms to produce toxins both the regulatory approval procedure will be much simpler and time and money can be saved in the production phase as the analytical program can be reduced.
Currently, there is a need for toxin-deficient Aspergillus mutant cells (i.e., safe organisms preferably classified as GRAS), which are suitable for producing polypeptides of interest in an efficient and economical way. The present invention satisfies this need by providing the production of polypeptides of interest using toxin-deficient Aspergillus mutant host cells and by providing a method to construct such mutant host cells. There is also a need for providing Aspergillus mutant cells, which in parent form habour (a) toxin gene(s), which is(are) not expressed, i.e., silent gene(s).
In accordance with one aspect of the present invention a method is provided for producing a polypeptide of interest by an Aspergillus mutant host cell, which comprises (a) cultivating a mutant of a parent Aspergillus cell, wherein (i) the mutant comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes responsible for the biosynthesis or secretion of at least one toxin, and (ii) the mutant produces less of the toxin than the parent Aspergillus cell when cultured under the same conditions; and (b) isolating the polypeptide from the culture medium.
In a preferred embodiment of the invention, the mutant Aspergillus cells produce at least about 90% less of the toxin than the parent cell when cultured under the same conditions. Preferably, the mutant produces less of a one or more of cyclopiazonic acid, kojic acid, 3-nitropropionic acid and aflatoxin than the parent Aspergillus cell when cultured under the same conditions.
According to another embodiment of the present invention toxin-deficient Aspergillus mutant host cells are provided useful for the production of a heterologous polypeptide of interest, which cell has been genetically modified in order to produce less of at least one toxin as compared to an Aspergillus parental cell, when cultured under the same conditions.
The Aspergillus mutant cells according to the invention are preferably selected from the group of A. oryzae, A. aculeatus, A. nidulans, A. ficuum, A. flavus, A. foetidus, A. soja, A. sake, A. niger, A. japonicus, A. parasiticus, and A. phoenicus. 
In a further embodiment of the present invention a method is provided for obtaining a toxin-deficient Aspergillus mutant host cell, which comprises (a) introducing into an Aspergillus parent host cell a first nucleic acid sequence encoding a polypeptide of interest and a second nucleic acid sequence comprising a modification of at least one of the genes responsible for the biosynthesis or secretion of at least one toxin; and (b) identifying the mutant from step (a) comprising the nucleic acid sequences.
In an aspect the invention relates to Aspergillus mutant cells, suitable for the expression of heterologous polypeptides, wherein one or more silent toxin genes have been eliminate.
These and other embodiments will be outlined in further detail in the description, which will follow hereinafter.